Protein phosphorylation controls a diverse array of biological processes. A series of exciting discoveries during the past few years has shown that protein-tyrosine phosphorylation plays a central role in intracellular signal transduction. Biochemical and molecular genetics studies of oncoproteins, growth factor response systems and cell-cycle control systems have uncovered an interconnected network of phosphorylation-mediated signaling pathways, but the biophysical mechanisms governing signal transduction are largely unknown. To elucidate the biophysical basis of various aspects of regulation, we are focusing on pp60c-src (Src), a cellular proto-oncoprotein and paradigm protein-tyrosine kinase. Upstream from its SH3 and SH2 domains, Src contains a "unique" domain which contains phosphorylation sites for at least three different kinases. Src is the best-studied representative of a family of nine proteins whose activity is regulated by multiple phosphorylation-dependent mechanisms. Two aspects of Src regulation will be addressed: 1) the mechanism of binding of its contiguous SH3-SH2 region to phosphotyrosine-containing target and regulatory peptides, and 2) the mechanism by which mitosis-specific phosphorylation in the unique domain modulates SH3-SH2 conformation, dynamics and ligand-binding affinity. Our initial project will investigate the role of the SH3 domain in stabilizing SH2/phosphopeptide interactions. A 20 kDa (180 residue) model protein containing both SH3 and SH2 domains (SH3-SH2) derived from chicken c-Src has been successfully overexpressed in E. coli, purified, and isotopically labeled with both 15N and 13C/15N. Two phosphopeptide ligands, a high affinity 11 residue peptide corresponding to the Src COOH terminus (modeling the intramolecular regulatory interaction), have been synthesized. The SH3-SH2 protein is stable at 1.2 mM, and preliminary spectra have been obtained in both the free and high-affinity phosphopeptide-bound forms. The 1H-15N HSQC spectrum of free 15N-SH3-SH2 shows a beautiful dispersion of peaks in both dimensions, with the expected number of cross peaks observed; a similar spectrum has been obtained for the ligand-bound form, with several changes in peak location. Examples of specific questions that will be studied are: Are there any long-lived intramolecular interactions between the SH3 and SH2 domains that contribute to phosphopeptide affinity? Does a head-to-tail intermolecular interaction occur in solution? What role do backbone dynamics play in phosphopeptide binding?